Support used in bioluminescent dosing of enzymes, substrates or enzymatic inhibitors

ABSTRACT

Method for the bioluminescent dosing of enzymes, substrates or enzymatic inhibitors, characterized in that there is used a transparent support made of synthetic plastic material previously treated with a charged hydrophilic substance for the Fixation by adsorption of an enzymatic system containing at least a luciferase. The invention extends to a support of the polystyrene type treated by a charged hydrophilic substance on which an enzymatic system containing at least a luciferase is fixed by adsorption.

The invention relates to an assay system consisting of a wall on whichis adsorbed at least one luciferase which, by acting consecutively withone or more adsorbed or unadsorbed enzymes, enables a series ofsubstrates, enzymes or enzyme inhibitors to be assayed bybioluminesence. This invention enables a series of biological moleculesto be assayed, and is hence applicable in biochemical analyses and inmedical and veterinary diagnosis.

Some luciferases are enzymes extracted from bacteria and which catalysethe reduction of flavin mononucleotide (hereinafter designated FMN) withthe concomitant production of photons. These photons can be detected andmeasured quantitatively in photometers or by any light-sensitivedetector. These luciferases can be combined with an NADH:FMNoxidoreductase or with an NADPH:FMN oxidoreductase (NADH=nicotinamideadenine dinucleotide in its reduced form; NADPH=nicotinamide adeninedinucleotide phosphate in its reduced form). Thus, it is possible, bymeans of these two enzymes working simultaneously, to assay NADH orNADPH in concentration regions ranging from 1 to 1000×10⁻¹² moles in thetest (Stanley, P. E. Methods in Enzymology, vol. 57, pages 215-222,1978; Lavi et al., J. Clin. Chem. Clin. Biochem., vol. 19, pages 749 andfollowing pages, 1981).

Another luciferase is isolated from fireflies, and enables photons to beproduced from the cleavage of adenosine triphosphate (ATP) to adenosinemonophosphate (AMP) and pyrophosphate.

Two developments of this method have been proposed: on the one hand, theinsolubilization of these two enzymes on agarose beads by coupling withcyanogen bromide, followed by the combination of these first two enzymeswith a third NADH- or NADPH-dependent dehydrogenase, which enables thesubstrate of this latter enzyme to be measured. The main achievementsare those of De Luca and his group, relating to the assay oftestosterone, L-malate, D-glucose, 6-P-gluconate, L-lactate, L-alanineand L-glutamate, with a minimal assay region ranging from 1.5 to10×10⁻¹² moles (U.S. Pat. No. 4,234,681 of M. A. De Luca-Mc Elroy;Weenhausen, G. and De Luca, M., Anal. Biochem., vol. 127, 380, 1982;Ford, J. and De Luca, M., Anal. Biochem., vol. 110, 43, 1981; Jablonski,E. and De Luca, M., Methods in Enzymology, vol. 57, 202, 1978). The 12biliary α-hydroxy acids can also be measured (Scholemerich et al., Anal.Biochem., vol. 133, 244, 1983). The method can also be used to measurethese dehydrogenases (Haggerty, C. et al., Anal. Biochem., vol. 88, 162,1978). While this system is sensitive and specific, it nevertheless hasthe drawbacks of introducing a solid support (agarose or the like) intothe reaction system, and this causes some retention of the emitted lightand is very sensitive to agitation at the time of measuring, since theuse of the porous support (agarose) requires the diffusion of thesubstrates into the meshes of the gel. One of the solutions proposed forthese problems is to pass the solution continuously through a cellcontaining the gel to which these enzymes are bound (Kricka et al.,Anal. Biochem., vol. 129, 392, 1983). This solution, while it deals withthe disadvantages mentioned above, involves making the assay techniquemore complex through the use of a special apparatus.

On the other hand, recently, V. Mookambeswaren and Sunanda (EuropeanPatent 0,169,767) have proposed covalent binding of luciferase to a gelmade from albumins linked to each other with glutaraldehyde.

The objective of the present invention is to remedy the disadvantages ofthe various systems described above, in the following manner: theinvention, as characterized in the claims, consists in adsorbing theenzymes on the walls of the tube which will be introduced into thephotometer, or on a strip of shape and size suitable to the measuringapparatus which can be a simple photographic film. This adsorption willbe accomplished via a layer of polylysine or of a copolymer ofpolylysine with another charged hydrophilic molecule. We havesuccessfully tried a copolymer of polylysine and polyphenylalanine, andobtained improved binding of luciferase. This increase in the adsorptionyield is explained by the dual hydrophilic and hydrophobic character ofthis copolymer, which will hence bind well to the wall of hydrophobicpolystyrene through its hydrophobic portion while maintaining thehydrophilic side towards the solution, on which wall the luciferase willbecome adsorbed. The adsorption yield is hence increased by using acopolymer of hydrophilic polylysine and another, more hydrophobic aminoacid. Other polymers of charged hydrophilic amino acids, such aspolyhistidine, a copolymer of polyhistidine, polyarginine or a copolymerof polyarginine could also replace the polylysine.

The advantages of this invention are to keep sensitivity and specificityof the luciferase system described above, but to avoid the problems ofretention of light and diffusion of substrate caused by the use of theporous support. In the case proposed here, the enzyme is immediately incontact with the reaction solution. Compared with the assay system in acontinuous-flow cell developed by Kricka et al. (above), our inventionhas the advantage of being simpler, of being immediately applicable tothe photometers currently on the market and of not having to take intoaccount the requirements imposed by continuous-flow assays: constantflow, suitably modified measuring cell, difficulty of large-scaleproduction.

Compared with the proposal of Mookambeswaren and Sunanda, our inventionmakes it possible to use immediately the support used for the assay(tube or strip) and to adsorb the enzyme directly on this support via ahydrophilic molecule. We avoid, on the one hand, the covalentmodification of the enzyme, which is especially sensitive to chemicalcoupling agents, and the support used is directly that which is used forthe detection apparatus, i.e. a tube for a photometer or a strip for theflat detector, and this enables a ready-to-use assay system to beproduced and greatly facilitates the industrial application.

The present invention consists in immobilizing, on the tube or stripwhich will be introduced into the photometer or placed in front of anyother apparatus for measuring photons, a luciferase with, if necessary,one of the NAD(P)H:FMN oxidoreductases and optionally one or more otherdehydrogenases or kinases. The tube coated in this manner can be usedfor assaying the substrate of the dehydrogenases or kinases, the enzymesthemselves or an inhibitor of these latter.

For this purpose, it is necessary to add in the requisite proportions,depending on the objective sought, the substrate of a dehydrogenase (orkinase), and NAD (or NADP or ADP), FMN (or nothing) and the substrate ofluciferase, usually decanal, these molecules usually being added in abuffer in the presence of molecules which stabilize the enzymes. As ageneral rule, the test molecule is introduced in a limiting amount withrespect to the other constituents of the system. The light emitted willbe measured and usually integrated during a given period, in order toincrease the accuracy of the assay.

There is a large number of NAD- or NADP-dependent dehydrogenases whichcan be used consecutively with the NAD(P)H:FMN oxidoreductase andluciferase; by way of example, we mention a few of these below:

lactose dehydrogenase

D-Glucose dehydrogenase

β-D-Galactose dehydrogenase

Glucose-6-phosphate dehydrogenase

Mannitol dehydrogenase

Mannitol-1-P dehydrogenase

Sorbitol dehydrogenase

Polyol dehydrogenase

Pentitol dehydrogenase

D-Xylitol dehydrogenase

2,3-cis-Polyol dehydrogenase

L-Threonic acid dehydrogenase

Lipoyl dehydrogenase

Lactate dehydrogenase

Glyoxylate dehydrogenase

Formaldehyde dehydrogenase

Formate dehydrogenase

Aldehyde dehydrogenase

Alcohol dehydrogenase

Acetaldehyde dehydrogenase

Fucose dehydrogenase

3-α-Hydroxysteroid dehydrogenase

β-Hydroxysteroid dehydrogenase

7-α-Hydroxysteroid dehydrogenase

3-α-20β-Hydroxysteroid dehydrogenase

We give below an example of assay of lactate dehydrogenase and oftestosterone using β-hydroxysteroid dehydrogenase. We have shown thatthe assay of this enzyme could be used for measuring the concentrationof an inhibitor of this enzyme, such as diethylstilboestrol (DES). Therelationship between the amount of DES and the percentage inhibition ofβ-hydroxysteroid dehydrogenase is not linear, but the assay of theinhibitor can nevertheless be accomplished by reference to a calibrationcurve produced with a standard DES preparation.

There is also a large number of kinases which can be used consecutivelywith the ATP-dependent luciferase. By way of example, we shall mention afew of these below:

Acetate kinase

Pyruvate kinase

Phosphoglycerate kinase

These kinases can be used co-immobilized with the luciferase, or free insolution or alternatively bound to another protein whose concentrationit is desired to measure. For example, the kinase can be measured whileit is bound to an antibody. This also applies to the dehydrogenasesmentioned above.

This list, while not exhaustive, shows the variety of substrates andenzymes which can be assayed by application of the method. The tubesprepared in the manner described can be lyophilized and stored for longperiods while retaining a substantial part of their activity.

EXAMPLE 1 Assay of NADH and NADPH

In this example, we describe in detail the insolubilization ofluciferase and of NADH:FMN oxidoreductase. This insolubilization wasoptimized in respect of various parameters: type of support, use ofpolylysine, concentration of poly-L-lysine, pH of binding of theenzymes, stabilization of the enzymes. In effect, the adsorption ofthese two enzymes serves as a basis for extending the discovery inconjunction with other dehydrogenases.

We used polystyrene tubes specially designed for their high adsorptionpower (Startube, NUNC, Roskilde, Denmark). The adsorption of thepoly-L-lysine was carried out by adding per tube 0.5 ml of poly-L-lysinesolution at a concentration of 80 μg/ml in a solution of 10 mmol/l NaCland 50 mmol/l Na phosphate, pH 8. The tubes are incubated for 1 hour at20° C. with a rotation of 5 revolutions per minute, and then rinsed 3times with 50 mmol/l Na phosphate buffer at pH 7.5. The enzyme solution(0.2 ml), containing 0.5 mg/ml of luciferase extracted from Vibrioharveyi (Sigma, St. Louis, Mo. L 1637) and 1 unit of NAD(P)H:FMNoxidoreductase extracted from Photobacterium fischeri (Sigma, cat.476-480) dissolved in 50 mmol/l Na phosphate buffer at pH 7.5 andcontaining 2 mmol/l of dithiothreitol, is then added to the tube andincubated for 30 min at 4° C. under rotation of 5 revolutions perminute. The tubes are rinsed twice in 10 mmol/l Na phosphate buffer atpH 7.5 containing 5 mg/ml of bovine serum albumin and 2 mmol/l ofdithiothreitol, and stored in this buffer.

For the assay, the reaction volume of 0.5 ml comprises 2.5 μmol/l ofFMN, 0.0005% of decanal, 2 mmol/l of dithiothreitol, 5 mg/ml of bovineserum albumin, and NADH in increasing concentrations in 10 mmol/l Naphosphate buffer at pH 7.5. The maximal emission of light was measured.It is found that this light emission is proportional to theconcentration of NADH present in the reaction medium for a concentrationregion ranging from 1 to 20,000×10⁻¹² moles in the test. The number ofphotons emitted per second at the maximum intensity ranges from 20 to10,000.

EXAMPLE 2 Assay of a dehydrogenase

In this example, we shall use the tubes on which the luciferase andNADH:FMN oxidoreductase have been adsorbed as described in Example 1,but they will be used for assaying lactate dehydrogenase in solution.For this purpose, the substrates will be added in excess such that theconcentration of the enzyme is the limiting factor. The reactionsolution of 0.5 ml contains 2.5 μmol/l of FMN, 0.0005% of decanal, 2mmol/l of dithiothreitol, 5 mg/ml of bovine serum albumin, 0.3 mmol/l ofNAD⁺ and 0.01 mol/l of L-lactate, and various concentrations of lactatedehydrogenase. The light emitted is measured and integrated during 60sec. The assay enables lactate dehydrogenase to be measured in aconcentration region ranging from 2×10⁻¹⁵ to 50×10⁻¹⁵ moles in the test.

EXAMPLE 3 Assay of the substrate of a dehydrogenase

In this example, the three enzymes luciferase, NADH:FMN oxidoreductaseand β-hydroxysteroid dehydrogenase will be immobilized on the tube inorder to carry out the assay of testosterone.

The adsorption of polylysine and the enzymes on the tube is carried outas described in Example 1, but the enzyme solution contains 0.075units/ml of β-hydroxysteroid dehydrogenase. For the assay oftestosterone, the tubes contain 0.5 ml of 10 mmol/l Na phosphate bufferat pH 7 and 25 μmol/l of FMN, 0.0001% of decanal, 5 mg/ml of bovineserum albumin, 2 mmol/l of dithiothreitol, 1.2 mmol/l of NAD and variousconcentrations of testosterone. The light intensity is measured andintegrated during 60 sec. The testosterone can be measured in aconcentration region ranging from 5×10⁻¹¹ to 5×10⁻⁹ moles in thereaction solution.

EXAMPLE 4 Assay of ATP by immobilization of ATP-dependent luciferase

The polystyrene tubes having a high adsorption power (Startube, NUNC,Roskilde, Denmark) were incubated overnight at 4° C. with a rotation of5 revolutions per minute for 30 min in the presence of a solution ofpoly(lysine-phenylalanine) at a concentration of 80 μg/ml in a solutionbuffered to pH 8 with 0.05 mol/l phosphate buffer. The tubes are thenrinsed twice with this same buffer. The solution (0.2 ml) of luciferaseextracted from Photinus pyralis (Sigma St Louis, Mo.) contains 40 μg/mland immunoglobulins G at a concentration of 0.1 mg/ml dissolved in 0.05mol/l Tris-acetate buffer, 60 μmol/l dithiothreitol, and is incubatedfor 30 minutes at 4° C. under rotation of 5 revolutions per minute. Thetubes are rinsed twice in the enzyme immobilization buffer.

For the assay, the reaction volume of 0.5 ml comprises 400 μl of 25mmol/l Tris-acetate buffer, pH 7.75, 75 μmol/l dithiothreitol, 125μmol/l EDTA, 6.25 mmol/l MgCl₂ and 7.5×10⁻⁵ mol/l luciferin.

The reaction is started with 100 μl of solution of increasingconcentrations of ATP. The light emission is proportional to theconcentration of ATP in a concentration region ranging from 1 to10,000×10⁻¹³ mol/l.

EXAMPLE 5 Assay of kinase

As an example of kinase assay, we used acetate kinase which catalysesthe following reaction:

    acetylphosphate+ADP→ATP+acetate

This enzyme can be extracted from thermophilic bacteria (Bacillusstearothermophilus) and is especially stable. The optimal conditions forbioluminescence assay of this kinase are as follows:

400 μl of 25 mmol/l Tris-acetate buffer, pH 7.75, 0.125 μmol/l EDTA, 75μmol/l dithiothreitol, 6.25 mmol/l MgCl₂, 7.5×10⁻⁵ mol/l luciferin, 1mmol/l acetylphosphate; the reaction is started with 10⁻⁶ mol/l ADP (100μl), the assay of the ATP produced being measured by the ATP-dependentluciferase immobilized on the polystyrene tube. Under these conditions,amounts as small as 10⁻¹⁷ and 10⁻¹⁸ μmol of kinase can be assayed inthis manner.

I claim:
 1. An immobilized enzyme device, comprising:a transparenthydrophobic plastic support; a layer of an amino acid polymer coatingthe support, said amino acid polymer having hydrophobic portions andhydrophilic portions and said amino acid polymer being oriented so thatthe hydrophobic portions of the polymer are bound to the support and thehydrophilic portions of the polymer extend outwardly from the support;and an enzyme composition adsorbed to the hydrophilic portions of thepolymer, said composition comprising a luciferase.
 2. The device ofclaim 1, wherein the transparent hydrophobic plastic substrate comprisespolystyrene.
 3. The device of claim 1, wherein the amino acid polymercomprises polylysine.
 4. The device of claim 1, wherein the amino acidpolymer comprises a copolymer of polylysine, polyarginine, orpolyhistidine and a hydrophobic amino acid polymer.
 5. The device ofclaim 4, wherein the amino acid polymer comprises a copolymer ofpolylysine and polyphenylalanine.
 6. The device of claim 1, wherein theenzyme composition further comprises NADH:FMN oxidoreductase orNADPH:FMN oxidoreductase.
 7. The device of claim 1 wherein the enzymecomposition further comprises a dehydrogenase or a kinase.
 8. A methodfor making an immobilized enzyme device comprising:coating a transparenthydrophobic plastic support with a layer of an amino acid polymer toform a pretreated support, said amino acid polymer having hydrophobicportions and having hydrophilic portions and amino acid polymer beingoriented so that the hydrophobic portions of the polymer are bound tothe hydrophobic plastic support and the hydrophilic portions of thepolymer extend outwardly from the plastic support; immobilizing anenzyme composition on the precoated support by adsorbing the enzymecomposition to the hydrophilic portions of the amino acid polymer, saidenzyme composition comprising a luciferase.
 9. The method of claim 8,further comprising the step of lyophilizing the immobilized enzymecomposition.
 10. An immobilized enzyme device made by the process ofclaim
 8. 11. The method of claim 8, wherein the transparent hydrophobicplastic substrate comprises polystyrene.
 12. The method of claim 8,wherein the amino acid polymer comprises polylysine.
 13. The method ofclaim 8, wherein the amino acid polymer comprises a copolymer ofpolylysine, polyarganine or polyhistidine and a hydrophobic amino acidpolymer.
 14. The method of claim 13, wherein the amino acid polymercomprises a copolymer of polylysine and polyphenylalanine.
 15. Themethod of claim 8, wherein the enzyme composition further comprisesNADH:FMN oxidoreductase or NADPH:FMN oxidoreductase.
 16. The method ofclaim 8, wherein the enzyme composition further comprises adehydrogenase or a kinase.
 17. A method for determining a test enzyme ina test solution, comprising:contacting an immobilized enzyme device witha test solution,said immobilized enzyme device comprises:a transparenthydrophobic plastic support; a layer of an amino acid polymer coatingthe support, said amino acid polymer having hydrophobic portions andhydrophilic portions and said amino acid polymer being oriented so thatthe hydrophobic portions of the polymer are bound to the support and thehydrophilic portions of the polymer extend outwardly from the support;and an enzyme composition adsorbed to the hydrophilic portions of thepolymer, said composition comprising a luciferase; and said testsolution comprising:the test enzyme and chemical species thatenzymatically react with the test enzyme and the luciferase to emitphotons; detecting photoemission from the contacted test solution; andquantifying the photoemission to determine the test enzyme in the testsolution.
 18. The method of claim 17, wherein the chemical speciescomprise a substrate of the test enzyme, a substrate of the luciferaseand ADP.
 19. The method of claim 17, wherein the test enzyme is akinase.
 20. The method of claim 17, wherein the enzyme compositionfurther comprises NADH:FMN oxidoreductase or NADPH:FMN oxidoreductase.21. The method of claim 20, wherein the chemical species comprise asubstrate of the test enzyme, a substrate of the luciferase, FMN and NADor NADP.
 22. The method of claim 17, wherein the test enzyme is adehydrogenase.
 23. The method of claim 17, wherein the test enzyme isconjugated with a protein and determining the test enzyme allowsdetermination of the protein.
 24. The method of claim 17, wherein thetest enzyme is conjugated with an antibody and determining the testenzyme allows determination of the antibody.
 25. A method fordetermining a substrate of a test enzyme, comprising:contacting animmobilized enzyme device with a test solution,said immobilized enzymedevice comprising:a transparent hydrophobic plastic support; a layer ofan amino acid polymer coating the support, said amino acid polymerhaving hydrophobic portions and hydrophilic portions and said amino acidpolymer being oriented so that the hydrophobic portions of the polymerare bound to the support and the hydrophilic portions of the polymerextend outwardly from the support; and an enzyme composition adsorbed tothe hydrophilic portions of the polymer, said composition comprising aluciferase and the test enzyme; and said test solution comprising: thesubstrate of the test enzyme, and chemical species that enzymaticallyreact with the test enzyme, and the luciferase to emit photons;detecting the photoemission from the contacted test solution; andquantifying the photoemission to determine the substrate of the testenzyme.
 26. The method of claim 25, wherein the chemical speciescomprise a substrate for the luciferase and ADP.
 27. The method of claim25, wherein the enzyme composition further comprises NADH:FMNoxidoreductase or NADPH:FMN oxidoreductase.
 28. The method of claim 25,wherein the chemical species comprise a substrate of the luciferase, FMNand NAD or NADP.
 29. A method for determining an inhibitor of a testenzyme, comprising:contacting an immobilized enzyme device with a testsolution,said immobilized enzyme device comprises:a transparenthydrophobic plastic support; a layer of an amino acid polymer coatingthe support, said amino acid polymer having hydrophobic portions andhydrophilic portions and said amino acid polymer being oriented so thatthe hydrophobic portions of the polymer are bound to the support and thehydrophilic portions of the polymer extend outwardly from the support;and an enzyme composition adsorbed to the hydrophilic portions of thepolymer, said enzyme composition comprising luciferase and the testenzyme, said test solution comprising: the inhibitor of the test enzymeand chemical species that enzymatically react with the test enzyme andthe luciferase to emit photons; detecting photoemission from thecontacted test solution; quantifying the photoemission; and determiningthe inhibitor by comparing photoemission by the contacted test solutionto photoemission by a standard solution comprising said chemical speciesand a known concentration of said inhibitor.
 30. The method of claim 29,wherein the chemical species comprise a substrate of the luciferase andADP.
 31. The method of claim 29, wherein the enzyme composition furthercomprises NADH:FMN oxidoreductase or NADPH:FMN oxidoreductase.
 32. Themethod of claim 31, wherein the chemical species further comprise asubstrate of the luciferase, FMN and NAD or NADP.
 33. A method fordetermining NADH or NADPH in a test solution, comprising:contacting animmobilized enzyme device with a test solution; said immobilized enzymedevice comprising:a transparent hydrophobic plastic support; a layer ofan amino acid polymer coating the support, said amino acid polymerhaving hydrophobic portions and hydrophilic portions and said amino acidpolymer being oriented so that the hydrophobic portions of the polymerare bound to the support and the hydrophilic portions of the polymerextend outwardly from the support; and an enzyme composition adsorbed tothe hydrophilic portions of the polymer, said composition comprising aluciferase and either NADH:FMN oxidoreductase or NADPH:FMNoxidoreductase; and said test solution comprising:NADH or NADPH, FMN anda substrate for the luciferase; and so as to emit photons detectingphotoemission from the contacted test solution; and quantifying thephotoemission to determine the NADH or NADPH in the test solution.
 34. Amethod for determining ATP in a test solution, comprising:contacting animmobilized enzyme device with a test solution,said immobilized enzymedevice comprising:a transparent hydrophobic plastic support; a layer ofan amino acid polymer coating the support, said amino acid polymerhaving hydrophobic portions and hydrophilic portions and said amino acidpolymer being oriented so that the hydrophobic portions of the polymerare bound to the support and the hydrophilic portions of the polymerextend outwardly from the support; and an enzyme composition adsorbed tothe hydrophilic portions of the polymer, said composition comprising aluciferase; and said test solution comprising ATP and a substrate forthe luciferase so as to emit photons, detecting photoemission from thecontacted test solution; and quantifying the photoemission to determinethe ATP in test solution.